脑钠肽单克隆抗体的制备及初步临床应用研究
发布时间：2019-02-16 21:32
【摘要】：目的: 心功能衰竭的早期诊断与干预在心脏疾患的诊治中非常重要。脑钠肽(BNP)作为一种主要来源于心室肌细胞的循环激素,在心室壁张力升高时迅速、大量分泌,其血浆浓度与心功能的相关性已被广泛认识。但目前国内检测BNP浓度靠进口国外的各种不同试剂盒,每种试剂盒检测范围及参考值不同,结果亦不尽相同,缺乏可比性,且价格昂贵。为使BNP检测国产化,本研究制备了BNP单克隆抗体,初步用于正常人及心脏病患者的血浆BNP浓度检测,以评价自制BNP单抗检测所得的BNP浓度与心功能的关系,为进一步研制国产BNP单抗试剂盒奠定基础。 方法: 1.根据GenBank中检索到的人BNP成熟蛋白序列,拼接BNP的基因。以原核表达系统pET32构建pET32a.BNP重组质粒,经诱导提取纯化,制备人Trx-BNP融合蛋白。 2.以Trx-BNP融合蛋白作抗原,免疫小鼠,获得分泌BNP抗体的杂交瘤细胞株。将杂交瘤细胞接种于小鼠腹腔,进行BNP单克隆抗体的大量制备。检测杂交瘤细胞的培养上清抗体效价及腹水抗体效价。以Western blot及免疫组化检测制备的BNP抗体特异性。 3.用制备的BNP单克隆抗体以ELISA间接法检测正常人及各级心功能的心脏病患者的血浆BNP浓度。正常人20例,心脏病患者92例,后者按纽约心脏病学会(NYHA)心功能分级分为:心功能Ⅰ级(24例)、Ⅱ级(23例)、Ⅲ级(20例)、Ⅳ级(25例),同时经超声心动图评价心脏收缩(EF值)及舒张功能(E/A值)。将心脏功能与BNP浓度作相关性分析,数据进行统计学处理。 结果: 1.利用原核表达系统pET32成功构建了pET32a.BNP重组质粒,并制备出高纯度的人Trx-BNP融合蛋白。 2.以Trx-BNP融合蛋白作抗原成功制备BNP单克隆抗体。抗体效价高,特异性强。
[Abstract]:Objective: the early diagnosis and intervention of heart failure is very important in the diagnosis and treatment of heart disease. Brain natriuretic peptide (BNP), as a circulating hormone mainly derived from ventricular myocytes, is secreted rapidly and in large quantities when ventricular wall tension increases. The relationship between plasma concentration and cardiac function has been widely recognized. However, at present, the detection of BNP concentration in China depends on various kinds of different kits imported from abroad. The range and reference value of each kit are different, the results are different, lack of comparability, and the price is expensive. In this study, monoclonal antibodies against BNP were prepared for the detection of plasma BNP in normal subjects and patients with heart disease in order to evaluate the relationship between the concentration of BNP and cardiac function. It lays a foundation for the further development of domestic BNP monoclonal antibody kit. Methods: 1. The gene of BNP was spliced according to the sequence of human BNP mature protein found in GenBank. PET32a.BNP recombinant plasmid was constructed by prokaryotic expression system (pET32). Human Trx-BNP fusion protein was prepared by extraction and purification. 2. The hybridoma cell lines secreting BNP antibody were obtained by immunizing mice with Trx-BNP fusion protein. Hybridoma cells were inoculated into mouse abdominal cavity to prepare monoclonal antibodies to BNP. The antibody titers of culture supernatant and ascites of hybridoma cells were detected. The specificity of BNP antibody was detected by Western blot and immunohistochemistry. 3. The BNP monoclonal antibody was used to detect the plasma BNP concentration in normal subjects and cardiac heart disease patients by ELISA indirect method. There were 20 normal persons and 92 patients with heart disease. According to the (NYHA) cardiac function classification of the New York Heart Association, the patients were divided into three groups: cardiac function grade 鈪，
本文编号：2424854
[Abstract]:Objective: the early diagnosis and intervention of heart failure is very important in the diagnosis and treatment of heart disease. Brain natriuretic peptide (BNP), as a circulating hormone mainly derived from ventricular myocytes, is secreted rapidly and in large quantities when ventricular wall tension increases. The relationship between plasma concentration and cardiac function has been widely recognized. However, at present, the detection of BNP concentration in China depends on various kinds of different kits imported from abroad. The range and reference value of each kit are different, the results are different, lack of comparability, and the price is expensive. In this study, monoclonal antibodies against BNP were prepared for the detection of plasma BNP in normal subjects and patients with heart disease in order to evaluate the relationship between the concentration of BNP and cardiac function. It lays a foundation for the further development of domestic BNP monoclonal antibody kit. Methods: 1. The gene of BNP was spliced according to the sequence of human BNP mature protein found in GenBank. PET32a.BNP recombinant plasmid was constructed by prokaryotic expression system (pET32). Human Trx-BNP fusion protein was prepared by extraction and purification. 2. The hybridoma cell lines secreting BNP antibody were obtained by immunizing mice with Trx-BNP fusion protein. Hybridoma cells were inoculated into mouse abdominal cavity to prepare monoclonal antibodies to BNP. The antibody titers of culture supernatant and ascites of hybridoma cells were detected. The specificity of BNP antibody was detected by Western blot and immunohistochemistry. 3. The BNP monoclonal antibody was used to detect the plasma BNP concentration in normal subjects and cardiac heart disease patients by ELISA indirect method. There were 20 normal persons and 92 patients with heart disease. According to the (NYHA) cardiac function classification of the New York Heart Association, the patients were divided into three groups: cardiac function grade 鈪，
本文编号：2424854
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